Mechanism of differential gene expression in alphavirus Arthropod borne (arbo) viruses are one of the largest groups of RNA viruses and are an important cause of endemic and epidemic outbreaks of encephalitis, hepatitis and hemorrhagic fevers around the world. Sindbis virus (SIN), the subject of this proposal, is a mosquito transmitted human alphavirus. Important mosquito transmitted viruses such as eastern equine encephalitis (EEE), western equine encephalitis (WEE) and venezuelan equine encephalitis (VEE) also belong to alphavirus group. All alphaviruses vary an 12kb long (+) sense RNA genome which is directly translated by host ribosomes to produce viral RNA polymerase. Conserved RNA motifs located at the termini of SIN genome are thought to be important for polymerase recognition and RNA synthesis. At least three different kinds of RNAs are expressed from SIN genome: i) 49S (+) sense RNA; II) 49s (-) sense RNA and iii) 26S (+) sense RNA. Although, differential expression of these RNAs is long known, the mechanism of regulation of their expression is lacking. We have recently characterized the minimal sequence requirements within the 3' AU-rich region which is necessary for RNA synthesis. Since, the first RNA to be made in SIN infected cells is the (-) sense SIN RNA, we have chosen first to address the mechanism of (-) sense RNA synthesis. On the basis of our own published results, and those of others, we hypothesize that one or more RNA motifs interact with cellular and viral factors to regulate RNA synthesis and differential gene expression. The specific aims of this proposal are: 1) To characterize the role of various SIN RNA motifs that regulate (-) sense RNA synthesis. 2) To map the compensatory nucleotide changes that accompany mutations within the 3'NTR of the SIN genome. These studies are new to alphaviruses and represent the beginning of our long term goal to delineate the differential regulation of alphaviral RNA synthesis. Since alphaviral RNA vectors are vigorously pursued as gene therapeutic and vaccine delivery modules, these proposed studies will be used in the development of improved RNA vectors.